Silica gel has unique properties which make it highly useful as a chromatographic support, and particularly applicable as a support for high-pressure liquid chromatography (HPLC). In the majority of its HPLC analytical applications, the material is used as a base for reversed-phase packing, which are used in a chromatographic mode in which aqueous organic solutions pass through a silica gel packing which has been modified by an alkylated silanizing agent, e.g. dimethyloctadecylchlorosilane. Even though the silanizing techniques and the silanizing materials themselves are similar, the resulting reversed-phase packings show a great variation in selectivity for various analytes, and particularly for silanophilic analytes. This variation results from the physical and chemical nature of the silica gel itself, and particularly variations in silica gels produced by different manufacturers.
Silica gel is the polymeric form of silicic acid, Si(OH).sub.4, in which siloxane bonds are formed between neighboring silicon atoms by eliminating water molecules. Wherever a break in the polymer structure occurs, including at the surface, a silanol group (Si--OH) is present. The surface density of silanol groups on the available HPLC silica gels is about 4.8 nm.sup.-2 or 8 .mu.mole/m.sup.2. These silanol groups react with monofunctional silylating reagents having the general formula XSi(CH).sub.3).sub.2 R, in which X is a good leaving group and R is a normal alkyl chain for most reverse-phase materials. Even with the most aggressive silanization reactions no more than half of the silanol groups can be converted to the alkyldimethylsilyl derivative; steric hinderance prevents a denser coverage of the surface. Thus a significant portion of the original silanol groups are left, and these interact with silanophilic analytes during chromatographic separations.
A useful solution to this problem of the residual silanol groups interacting with the analytes is to saturate the residual silanols with another silanophile added as a component of the mobile phase. By choosing the appropriate base additive, sharply eluting bands can be obtained for such analytes. Similarly, a cetyltrimethylammonium bromide can be used under certain conditions in a reversed-phase mode even with unmodified silica gel. In this case the adsorption of the silanophilic "head", the quaternary amine, forms a reversed-phase layer of its lipophilic "tail", thus becoming a competing reagent and a lipophilic layer at the same time.
A more permanent solution to the problem is encapsulation of the silica support. Nonpolar linear polymers may be adsorbed onto the silica surface, followed by gamma-ray irradiation to initiate crosslinking. This yields a permanent, nonextractable coating. Such encapsulated silica or alumina supports show high efficiency and resolution for basic silanophilic compounds. Shiseido Company of Japan held encapsulation to be responsible for the superior resolution they report having observed for basic amino analytes on its S/S-C18 reversed-phase packing. Thus covering the surface of the silica gel, in the first case by a nonbonded polymeric network, and in the second case by a bonded polymeric network, can prevent the deleterious effect of the residual silanol groups.
Glajch et al., in U.S. Pat. No. 4,705,725, disclose development of sterically protected, bonded organosilanes to prevent residual silanols from interacting with silanophilic analytes, as for example in --Si(i-Pr).sub.2 R, where the two bulky isopropyl (i-Pr) residues render the residual silanols less accessible to such analytes.
Also known to those skilled in the art is that on highly polar, bonded silica-based phases, such as propylamino and propylcyano phases, good peak shapes and efficiencies for separations of silanophiles may be obtained. These polar phases consist of silica gel modified with 3-amino or 3-cyanopropyltrimethoxysilane. By shortening the alkyl chain of the bonded ligand, the lipophilic character of the supports diminishes and its polar character, including the contribution from the underlying silanol groups, predominates. Because of competitive interactions of the bonded polar groups with the silanols and the high polarity of the phase, the otherwise sluggish adsorption/desorption kinetics of the basic analytes is enhanced. Similarly, very polar polymers strongly adsorbed onto the silica surface, such as polyethyleneimine crosslinked with an organic diepoxide and partially modified with short-chain acids, also show good chromatographic efficiency for basic compounds. These polymers encapsulate and deactivate the silanols through the basic nitrogens of the network.
Modification of bonded .gamma.-aminopropyl groups by acyl chlorides, active esters, or isocyanates is well documented, as is modification of 3-aminopropyltrialkoxysilane, followed by bonding of the resulting modified product to a silica gel support. Such modifications were done primarily as a convenient synthetic tool to functionalize a bonded silica surface, as for example, to prepare a chiral stationary phase, a .pi.--.pi. complexing surface, and the like, or when the alternative synthetic route to a product via hydrosilylation of an olefinic intermediate was difficult. The only disclosure known to the applicant of studying reversed-phase acylations was by Nomura et al (Analytical Sciences, Vol. 3(1987), pages 209-212), and was intended to determine the effect of the molecular size of the acylating reagents on acylation of aminopropyl ligands bonded to silica gels having different pore diameters.